Genetic interactions between Polycystin-1 and TAZ in osteoblasts define a novel mechanosensing mechanism regulating bone formation in mice

Molecular mechanisms transducing physical forces in the bone microenvironment to regulate bone mass are poorly understood. Here, we used mouse genetics, mechanical loading, and pharmacological approaches to test the possibility that polycystin-1 and TAZ have interdependent mechanosensing functions in osteoblasts. We created and compared the skeletal phenotypes of control Pkd1flox/+;TAZflox/+, single Pkd1Oc-cKO, single TAZOc-cKO, and double Pkd1/TAZOc-cKO mice to investigate genetic interactions. Consistent with an interaction between polycystins and TAZ in bone in vivo, double Pkd1/TAZOc-cKO mice exhibited greater reductions of BMD and periosteal MAR than either single TAZOc-cKO or Pkd1Oc-cKO mice. Micro-CT 3D image analysis indicated that the reduction in bone mass was due to greater loss in both trabecular bone volume and cortical bone thickness in double Pkd1/TAZOc-cKO mice compared to either single Pkd1Oc-cKO or TAZOc-cKO mice. Double Pkd1/TAZOc-cKO mice also displayed additive reductions in mechanosensing and osteogenic gene expression profiles in bone compared to single Pkd1Oc-cKO or TAZOc-cKO mice. Moreover, we found that double Pkd1/TAZOc-cKO mice exhibited impaired responses to tibia mechanical loading in vivo and attenuation of load-induced mechanosensing gene expression compared to control mice. Finally, control mice treated with a small molecule mechanomimetic MS2 had marked increases in femoral BMD and periosteal MAR compared to vehicle control. In contrast, double Pkd1/TAZOc-cKO mice were resistant to the anabolic effects of MS2 that activates the polycystin signaling complex. These findings suggest that PC1 and TAZ form an anabolic mechanotransduction signaling complex that responds to mechanical loading and serve as a potential novel therapeutic target for treating osteoporosis.

Pkd1/TAZ Oc-cKO mice to investigate genetic interactions. Consistent with an interaction between 23 polycystins and TAZ in bone in vivo, double Pkd1/TAZ Oc-cKO mice exhibited greater reductions of 24 BMD and periosteal MAR than either single TAZ Oc-cKO or Pkd1 Oc-cKO mice. Micro-CT 3D image 25 analysis indicated that the reduction in bone mass was due to greater loss in both trabecular bone 26 volume and cortical bone thickness in double Pkd1/TAZ Oc-cKO mice compared to either single 27 Pkd1 Oc-cKO or TAZ Oc-cKO mice. Double Pkd1/TAZ Oc-cKO mice also displayed additive reductions in 28 mechanosensing and osteogenic gene expression profiles in bone compared to single Pkd1  or TAZ Oc-cKO mice. Moreover, we found that double Pkd1/TAZ Oc-cKO mice exhibited impaired 30 responses to tibia mechanical loading in vivo and attenuation of load-induced mechanosensing 31 gene expression compared to control mice. Finally, control mice treated with a small molecule 32 mechanomimetic MS2 had marked increases in femoral BMD and periosteal MAR compared to 33

Introduction 38
In vivo and in vitro studies demonstrate that the polycystin-1(PC1)/polycystin-2 (PC2) 39 heterotrimeric complex functions in osteoblasts and osteocytes to regulate bone mass 1,2 and acts 40 as a mechanosensor to transduce the bone anabolic response to mechanical loading in vivo 3,4 . 41 Genetic ablation of either PC1 or PC2 deficiency in osteoblasts or osteocytes has similar effects 42 in reducing bone mass by decreasing osteoblast-mediated bone formation 3,5-8 . PC1 and PC2 43 mechanosensing functions are mediated by heterotrimeric complex activation of common signal 44 transduction pathways. In this regard, PC1 and PC2 conditional knockout models exhibit 45 concordant effects in impairing osteoblast-mediated bone formation. However, PC1 and PC2 46 have discordant effects on bone marrow adipogenesis that implicates separate signaling 47 mechanism 4,6,9 . In this regard, PC1 deficiency stimulates adipogenesis, leading to increased 48 bone marrow adipose tissue (MAT) deposition 2,6 , whereas PC2 loss-of-function inhibits 49 adipogenesis 4 . Additional in vitro and in vivo data show that PC1 activates Runx2 transcriptional 50 activity to stimulate osteoblastogenesis but diminishes PPARγ signaling leading to reduced bone 51 marrow fat 2,4 . In agreement with the low turnover bone disorder in Pkd1 mouse models, the blood 52 level of bone-specific alkaline phosphatase was significantly lower in patients with ADPKD 53 compared to control subjects without ADPKD 10-12 . The molecular mechanisms underlying the 54 different effects of PC1 and PC2 on osteoblastogenesis and adipogenesis are not clear. In the 55 current studies, we sought to understand the mechanism for the apparent PC1-specific effect in 56 reciprocally regulating transcriptional control of osteoblastogenesis and adipogenesis. 57 The Hippo-YAP/TAZ pathway is also regulated by mechanical forces [13][14][15][16] . Alterations of 58 matrix stiffness in cell culture modulates nuclear translocation of non-phosphorylated TAZ 59 resulting in co-activation of Runx2 and stimulation of osteoblastogenesis and in TAZ binding to 60 PPARγ to inhibit adipogenesis 2,17,18 . The physiological importance of TAZ in bone homeostasis 61 is revealed by transgenic overexpression of TAZ in osteoblasts in mice, which increases 62 osteoblast-mediated bone formation and inhibits bone marrow adipogenesis 19 ; depletion of TAZ in zebrafish, which impairs bone development 18 , and global knockout of TAZ in mice, which 64 have small stature and ossification defects 20 . Based on these observations, we posit that PC1 65 dependent TAZ signaling might explain the differential functions of PC1 and PC2 on 66

adipogenesis. 67
Recent studies show crosstalk between PC1 and TAZ signaling that is mediated by the 68 binding of TAZ to the PC1 C-terminal tail 2,17 . The PC1/TAZ complex is cleaved to allow nuclear 69 translocation of TAZ, a mechanism of TAZ regulation that differs from the canonical Hippo 70 regulation of YAP/TAZ signaling 21 . TAZ binds to the PC1-CTT and undergoes nuclear 71 translocation in response to changes in bone ECM microenvironment to stimulate 72 osteoblastogenesis and inhibit adipogenesis through transcriptional co-activation of Runx2 and 73 co-repression of PPARγ activity 2 . TAZ also binds to PC2, leading to PC2 degradation 22 . These 74 observations in bone parallel the interactions between PC1/PC2 and TAZ in primary cilia in renal 75 epithelial cells 20,23,24 . In this regard, TAZ knockout from the kidney result in cystic kidney disease 76 in mice, similar to polycystin complex inactivation, suggesting that PC1/PC2 and TAZ act through 77 common pathways in the kidney as well as bone 20,23,24 . Furthermore, a small molecule 78 mechanomimetic (named as molecular staple, MS) that binds to the PC1:PC2 C-terminal tail in a 79 presumptive coiled-coil region (e.g. PC1 residue Tyr 4236 and PC2 residues Arg 877 , Arg 878 , and 80 Lys 874 ) has been shown to activate this complex and mimic the effects of physical forces to 81 activate polycystins/TAZ signaling and stimulate bone mass in mice 2 . Collectively these 82 observations suggest that TAZ modulates polycystin's mechanosensing functions to differentially 83 regulate osteoblastogenesis and adipogenesis. 84 In this study, we examined the interaction between PC1 and TAZ in mouse bone by using 85 Osteocalcin (Oc)-Cre to conditionally delete both Pkd1 and TAZ in osteoblasts. We compared 86 skeletal phenotypes of double Pkd1/TAZ Oc-cKO mice with single conditional Pkd1 and TAZ null 87 mice under baseline conditions, after mechanical loading, and following treatment with a more 88 potent mechanomimetic, MS2, that activates the PC1/PC2 complex. We found that genetic ablation of PC1 and TAZ in osteoblasts results in additive loss of bone mass and anabolic 90 responses to mechanical loading. Compound PC1 and TAZ deficient mice were also resistant to 91 the bone inductive effects of the MS2 mechanomimetic in vivo. Our findings provide a new 92 mechanism whereby TAZ regulates skeletal homeostasis through co-dependent functions with 93 PC1 in osteoblasts. 94 well as YAP signaling such as increased CYR61 and CTGF gene expressions inhibits osteoblast 121 differentiation and mineralization. An increase in RankL and the RankL/OPG ratio promotes 122 osteoclast differentiation, leading to greater TRAP staining and higher osteoclast activity in 123 conditional TAZ Oc-cKO null mice compared with control mice (TAZ +/+ ). Conditional deletion of TAZ 124 also resulted in increased adipocyte markers such as PPARγ2, aP2 and Lpl gene expressions 125 (Table 1). 126 Unexpectedly, global TAZ +/heterozygous mice, which had a ~40% reduction in TAZ 127 message expression in bone, did not have significant changes in BMD or bone volume. Single-128 heterozygous TAZ +/showed normal bone gene expression profiles as well ( Table 1) Micro-CT 3D image analysis and Goldner staining (Fig 3 & 4). Also, the reductions in bone mass 142 were similar in male and female adult mice. 143 The skeletal phenotype of double Pkd1/TAZ Oc-cKO mice was more severe than either single 144 Pkd1 Oc-cKO or TAZ Oc-cKO null mice. Double Pkd1/TAZ Oc-cKO mice had greater losses in BMD, 145 trabecular bone volume, and cortical bone thickness with 33%, 53%, and 27% reductions, respectively in both male and female adult mice. This indicates the additive effects of Pkd1 and 147 TAZ in postnatal bone homeostasis (Fig 3 & 4). Consistent with lower bone mass, combined Pkd1 148 and TAZ deficiency also resulted in additive reductions in osteoblast-related gene expressions, 149 such as in Runx2-II, Osteocalcin, and Dmp1 (Table 2), as well as mechanosensing responsive 150 genes such as in Wnt10b, c-Jun, and PTGS2 ( Table 2). Periosteal MAR (Fig 4) was decreased 151 by ~73% in conditional double Pkd1/TAZ Oc-cKO mice compared to controls, whereas TAZ Oc-cKO and 152 Pkd1 Oc-cKO single conditional knockout mice had reductions in periosteal MAR of 55% and 53%, 153 respectively compared to control mice. Loss of either Pkd1 or TAZ resulted in enhanced marrow 154 adipogenesis, but no additive effects on adipocyte differentiation-related gene expressions were 155 observed in the conditional double Pkd1/TAZ Oc-cKO mice ( Table 2). 156 We found that the conditional deletion of Pkd1 or TAZ in osteoblasts has opposite effects 157 on osteoclast activities. There was a decreased RankL/OPG expression ratio and TRAP 158 immunostaining in Pkd1 Oc-cKO mice but an increased RankL/OPG expression ratio and TRAP 159 immunostaining in TAZ Oc-cKO mice ( Table 2 and Fig 4). In contrast, double Pkd1/TAZ Oc-cKO had 160 similar RankL expression and TRAP immunostaining when compared to control, indicating a 161 recovery of osteoclast activities in the double null mice ( Table 2 and Fig 4). Changes in gene 162 expression and immunostaining in bone correlated with alterations in serum biomarkers (Table  163 3). In this regard, further evidence for osteoblast and osteocyte dysfunction includes reductions 164 in serum osteocalcin and FGF-23 from in single Pkd1 Oc-cKO or TAZ Oc-cKO mice compared with age-165 matched control mice and an even greater decrement in double Pkd1/TAZ Oc-cKO null mice (Table  166 3). In contrast, serum levels of TRAP, a marker of bone resorption, were decreased in single 167 Pkd1 Oc-cKO mice, increased in single TAZ Oc-cKO mice, but restored in double Pkd1/TAZ Oc-cKO null 168 mice compared with control littermates ( Table 3). In addition, serum level of leptin was 169 significantly higher in single Pkd1 Oc-cKO or TAZ Oc-cKO mice than age-matched control mice. 170 However, we did not observe further increase in double Pkd1/TAZ Oc-cKO null mice (Table 3). These findings suggest that Pkd1 and TAZ have distinct functions among osteoblasts, adipocytes, and 172 osteoclasts in bone in vivo. 173

Loss of mechanical loading response in conditional Pkd1 and TAZ deficient mice. 174
The cross-sections of tibiae from control and double Pkd1/TAZ Oc-cKO null mice after 175 mechanical tibia loading studies in vivo are shown in Fig 5. In wild-type control mice, loaded tibia 176 showed a 2-fold increase in periosteal mineral apposition rate. In contrast, there was no 177 measurable increase in periosteal mineral apposition in the loaded tibia from double Pkd1/TAZ 178 knockout mice (Fig 5). In addition, a real-time RT-PCR analysis revealed that loaded tibia from 179 the control mice had a dramatic response to mechanical stimulation, evidenced by significant 180 increases of mechanosensing and osteogenic gene expressions including Wnt10b, FzD2, Axin2, 181 PTGS2, c-Jun, c-Fos, Runx2-II, Osteocalcin, ALP, and Dmp1 when compared with no load control 182 tibia. In contrast, even when using the same loading regimen, no changes of mechanosensing 183 and osteogenic gene expression profiles were observed in the loaded tibia from double 184 Pkd1/TAZ Oc-cKO null mice when compared with no load control tibia (Table 4). Thus, PC1 and TAZ 185 are important in mediating mechanotransduction in bone. 186

Validation of MS2 key binding to residues in PC1/PC2 C-terminus tails. 187
We have previously showed that the small molecule MS2 activates PC1/PC2 complex 188 signaling 2 . Using computational modeling, we engaged in an induced fit docking campaign and 189 predicted several potential ligand binding complexes. From these predicted poses, we identified 190 key residues in the PC1 and PC2 C-terminus tail regions with which MS2 is predicted to interact. 191 As shown in Fig 6, the PC2-CTT binding site for MS2 is predicted to include Lysine 874 and 192 Arginine 877, whereas the PC1-CTT binding site for MS2 involves Tyrosine 4236 (Fig 6A & 6B). 193 To test these predictions, we performed site-mutagenesis of key residues in both PC1 and PC2 194 and tested the effects of MS2 on PC1 and PC2 assembly using a BRET 2 assay. We observed 195 that the compound MS2 markedly enhances BRET 2 signal in wild-type constructs, while 196 mutagenesis of key residues in either PC1-CTT or PC2-CTT constructs completely abolished the BRET 2 signal in the presence of compound MS2, confirming a role of MS2 in binding and 198 enhancing the PC1 and PC2 interaction (Fig 6C & 6D). 199 Next, we examined PC1/PC2 complex formation during MC3T3-E1 osteoblast 200 differentiation in vitro. We observed culture duration dependent increase of PC1/PC2 complex 201 formation by western blot analysis. Incubation with 1 µM of MS2 in osteogenic cultures markedly 202 increased the amount of PC1 and PC2 protein as assessed by western blot analysis (Fig 6E &  203   6F). These data suggests that MS2 may molecularly stabilize the PC1/PC2 complex in osteoblast 204 culture in vitro. 205

Loss of MS2-mediated stimulated increase in bone mass in conditional Pkd1 and TAZ 206 deficiency mice. 207
Finally, we treated wild-type and conditional double Pkd1/TAZ Oc-cKO null mice with vehicle 208 or MS2 (50 mg/kg) i.p. daily and assessed their skeletal response. After only 2 weeks, we 209 observed that wild-type control mice treated with MS2 had a 15% increment in femoral bone 210 mineral density compared to vehicle control (Fig 7). Micro-CT 3D images revealed that MS2 211 treated wild-type mice had a 39% increase in trabecular bone volume and 16% increase in cortical 212 bone thickness. 213 In contrast, administration of MS2 had no effects on bone mineral density and bone 214 structure in double Pkd1/TAZ Oc-cKO null mice, suggesting specific-target dependent effects of MS2 215 on polycystins/TAZ signaling (Fig 7). We also observed that there were 1.6-fold increases in bone 216 formation rate in wild-type mice treated with MS2 compared to the vehicle control, in agreement 217 with enhanced osteoblastogenesis (e.g., Runx2-II, OOsteocalcin, ALP and Dmp1) and 218 suppressed marrow adipogenesis (e.g., PPARγ2, aP2, and Lpl) by a real-time RT-PCR analysis 219 (Table 5 and Fig 7). Again, administration of MS2 had no effects on bone formation rate and 220 bone gene expression profiles in double Pkd1/TAZ Oc-cKO null mice. Furthermore, MS2 stimulated 221 mechanosensing gene expressions, including Wnt1, Wnt10b, FzD2,eNOS,222 and PTGS2, consistent with MS2 acting as a small molecule "mechanomimetic". MS2 treatment decreased RankL/OPG expression ratio and TRAP immunostained osteoclasts in the MS2-224 treated mice compared to vehicle control (Table 5 and Fig 7). Administration of MS2 had no 225 effects on osteoblast-mediated bone formation rate, marrow adipogenesis, and osteoclast activity 226 in conditional double Pkd1/TAZ Oc-cKO null mice (Table 5 and Fig 7). These data support that MS2 227 functions as anabolic drugs through the polycystins/TAZ pathway to promote the bone remodeling 228

process. 229 230
Discussion 231 In the current study, we provide loss-of-function genetic and gain-of-function 232 pharmacological evidence for the co-dependent roles of PC1 and TAZ in regulating osteoblast-233 mediated bone formation and bone mass, First, using Oc-Cre to conditionally delete Pkd1 and 234 TAZ in osteoblasts in mice, we found that deletion of both genes in double Pkd1/TAZ Oc-cKO null 235 mice resulted in a more severe skeletal phenotype than loss of either PC1 or TAZ alone in the Osteocytes regulate osteoclast activity through the RANKL/OPG paracrine pathway 36,37 . 300 no difference in OPG expression in the Pkd1 Oc-cKO null and TAZ Oc-cKO null mice, this could account 302 for the differential effects of PC1 and TAZ on osteoclast activity in bone. Moreover, MS2 303 significantly decreased RANKL expression in bone and attenuated osteoclast activity. Our 304 understanding of TAZ regulation of osteoclast function is further supported by the observation by 305 Yang et al that either global or osteoclast-specific knockout of TAZ led to a low-bone mass 306 phenotype due to elevated osteoclast formation 38 . Thus, PC1 and TAZ signaling have divergent 307 effects on osteoclasts and bone resorption. 308 Finally, a strong association exists in senile osteoporosis between decreased 309 osteoblastogenesis and increased adipogenesis leading to increased bone marrow fat 39-42 . We 310 have previously reported that global PC1 deficiency in mice has an inverse effect, inhibiting 311 osteoblastogenesis and stimulating adipogenesis 2,7 . Similar to conditional Pkd1 Oc-cKO null mice 312 3,6 , we also observed conditional TAZ Oc-cKO null mice had greater increments in adipogenic 313 markers than global or conditional TAZ heterozygous mice in the current study, suggesting a 314 gene-dosage dependent effect of loss-of-TAZ in osteoblasts on bone marrow adipogenesis. 315 Interestingly, we found a similar increase in adipogenic markers in both PC1 and/or TAZ 316 osteoblast conditional knockout mice. The increase of adipogenic markers could be theoretically 317 explained by increased transdifferentiation of osteoblast precursors to adipocytes 43,44 , or effects 318 of PC1 and TAZ in osteoblasts/osteocytes differentially releasing paracrine factors that modulate 319 adipogenesis 45-47 , analogous to paracrine factors that regulate osteoclastogenesis. Regardless, 320 our studies revealed that double Pkd1/TAZ Oc-cKO null mice had no differences in adipogenic 321 markers relative to single Pkd1 Oc-cKO or TAZ Oc-cKO null mice, suggesting that polycystin-1 and TAZ 322 regulate adipocyte differentiation through the common polycystins/TAZ pathway. to determine bone volume (BV/TV) and cortical thickness (Ct.Th) as previously described 3,4,6 . 359

Real-time quantitative reverse transcription PCR (real-time qRT-PCR) and western blot 360
analysis. For real-time qRT−PCR, 1.0 g total RNA isolated from either the long bone of 6-week-361 old mice or 8-days cultured BMSCs in differentiation media was reverse transcribed as previously 362 described 4,6 . PCR reactions contained 20 ng template (cDNA), 375 nM each forward and reverse 363 primers, and 1 X EvaGreen Supermix (Bio-Rad, Hercules, CA) in 10 l. The threshold cycle (Ct) 364 of tested-gene product from the indicated genotype was normalized to the Ct for cyclophilin A. 365 Then the tested-gene product vs cyclophilin A is normalized to the mean ratio of wild-type or 366 control group, which has been set to 1. 367 For Western blot analysis, protein concentrations of the supernatant were determined with 368 a total protein assay kit (Bio-Rad, Hercules, CA). Equal quantities of protein were subjected to 4-369 12% Bis-Tris or 3-8% Tris-Acetate gradient Gels (Invitrogen, Carlsbad, CA) and were analyzed 370 with standard western blot protocols as previously described 4,6 . Polycystin-1 antibody (7E12, sc- were from Santa Cruz Biotechnology (Paso Robles, CA). The intensity of the bands was 376 quantified using Image J software (http://rsb.info.nih.gov/ij/). National Laboratory and The University of Tennessee, Knoxville, we previously identified a 379 compound that is thought to bind to the polycystin1 (PC1-CTT)/polycystin2 (PC2-CTT) complex 380 in their C-terminus tails that we refer to as molecular staple two (MS2) 2 . Here, using computational 381 ligand docking with an initial rigid receptor search using the proxy triangle algorithm and London 382 dG scoring function and subsequent induced-fit refinement using a "free" receptor geometry, the 383 were plated in 96-well black isoplate and cultured for 48 hours after transfection. We used the 397 Synergy H4 plate reader to monitor the BRET 2 signal (Fluorescence/Luminescence ratio). The 398 relative fluorescence (515/30 nm) and luminescence (410/80 nm) raw data were detected from 399 each well after adding DeepBlue C (5 µM) in the presence or absence of compound MS2 (10 µM). 400 In addition, based on the identification of crucial contact residues [e.g. Lys(K) 874    structure by micro-CT 3D images analysis from male mice. Data are expressed as the mean ± 595 S.D. from serum samples of individual mice (n=6). *P < 0.05, **P < 0.01, ***P < 0.001 compared 596 with wild-type mice, ## P < 0.01 compared with, TAZ Oc-cKO mice, and && P < 0.01 compared with Pkd1 Oc-cKO mice, respectively. P values were determined by 1-way ANOVA with Tukey's multiple-598 comparisons test. conditional Pkd1 and/or TAZ deleted mice compared with age-matched control mice. c Periosteal 604 mineral apposition rate (MAR) by Calcein double labeling. There was a significant reduction in 605 periosteal MAR in single Pkd1 Oc-cKO or TAZ Oc-cKO mice compared with age-matched control mice 606 and an even greater decrement in double Pkd1/TAZ Oc-cKO null mice, indicating a synergic effect 607 of PC1 and TAZ on osteoblast-mediated bone formation. d TRAP staining (red color) for 608 osteoclast activity. Data are expressed as the mean ± S.D. from 6 individual mice (n=6). *P < 609 0.05, **P < 0.01, ***P < 0.001 compared with wild-type mice. P values were determined by 1-way 610 ANOVA with Tukey's multiple-comparisons test. Data are mean  S.D. from 6 tibias of wild-type control and compound Pkd1/TAZ Oc-cKO null mice. 635 *P < 0.05, **P < 0.01, ***P < 0.001 compared with wild-type control mice. P values were 636 determined by 1-way ANOVA with Tukey's multiple-comparisons test. 637 Table 5. Gene expression profiles in bone from MS2-treated wild-type control and